Atypically Shaped Setae in Gall Mites (Acariformes, Eriophyoidea) and Mitogenomics of the Genus Leipothrix Keifer (Eriophyidae)

Simple Summary Eriophyoidea (gall mites) are a megadiverse lineage of worm-like mites that feed on vascular plants. The setae of these mites are sometimes distinctive because of their atypical shape, either bifurcated, angled or swollen, but never many-branched. Our study of eriophyoid setae revealed that bifurcated and angled setae are widely distributed across Eriophyoidea. The group of worm-like soil mites (Nematalycidae) with which they are affiliated have bifurcated and trifurcated setae. The plesiomorphic and also most common state of all mites is hyper-furcating setae (more than three branches), which are almost always represented in rows (seri-furcating). The likely explanation for the filiform and unbranched setae of Eriophyoidea is the gradual loss of branches, one by one, with the bifurcated state that is shared with Nematalycidae being an ancestral state. Angled setae are an intermediary state because they probably represent a bifurcating seta with only a single branch; the other one is completely diminished. Accordingly, hypo-furcating setae (three or fewer branches) are a synapomorphy that unites Eriophyoidea with Nematalycidae. Our phylogenetic analyses also showed that Leipothrix, the largest genus with a bifurcated seta on the palps, is monophyletic once Cereusacarus juniperensis is excluded and five species of Epitrimerus have been transferred into this genus. Abstract The setae in Eriophyoidea are filiform, slightly bent and thickened near the base. Confocal microscopy indicates that their proximal and distal parts differ in light reflection and autofluorescence. Approximately 50 genera have atypically shaped setae: bifurcated, angled or swollen. These modifications are known in the basal part of prosomal setae u′, ft′, ft″, d, v, bv, ve, sc and caudal setae h2. We assessed the distribution of atypically shaped setae in Eriophyoidea and showed that they are scattered in different phylogenetic lineages. We hypothesized that the ancestral setae of eriophyoid mites were bifurcated before later simplifying into filiform setae. We also proposed that hypo-furcating setae are a synapomorphy that unites Eriophyoidea with Nematalycidae. We analyzed four new mitochondrial genomes of Leipothrix, the largest genus with bifurcated d, and showed that it is monophyletic and has a unique mitochondrial gene order with translocated trnK. We exclude Cereusacarus juniperensis n. comb. Xue and Yin, 2020 from Leipothrix and transfer five Epitrimerus spp. to Leipothrix: L. aegopodii (Liro 1941) n. comb., L. femoralis (Liro 1941) n. comb., L. geranii (Liro 1941) n. comb., L. ranunculi (Liro 1941) n. comb., and L. triquetra (Meyer 1990) n. comb.


Introduction
The superfamily Eriophyoidea (gall mites or four-legged mites) is a lineage of highly host-specific, permanent parasites of higher vascular plants.They have an unusual morphology for acariform mites, based on an elongate, vermiform body and only two pairs of legs.Eriophyoidea have moved to a completely different position within the system of Acariformes in recent years.For a long time, they were considered members of the cohort Eupodina, within the order Trombidiformes [1][2][3].However, morphological and molecular phylogenetic studies performed in the last five years indicate that eriophyoids do not belong to Trombidiformes, placing them instead in a notably more basal position with soil mites of the family Nematalycidae, near the root of the Acariformes tree [4].This long-term misinterpretation of Eriophyoidea was largely because of a number of homoplasies that were attributed too much importance, including plant feeding and a suite of interdependent paedomorphisms [2,4,5].
Reduced morphology, simplification, microscopic size and deficiency of phylogenetically informative characters are inherent characteristics of gall mites, affecting their systematics [1,2,[6][7][8].A large set of characters has been developed for classifying the supraspecific taxa of Eriophyoidea during the 20th century [9][10][11][12].It includes such groups of traits as the following: chaetome (the set of all setae), position and shapes of setal tubercles, segmentation of legs, structure of the gnathosoma and female internal genitalia, and various characteristics of the opisthosomal cuticle, including the shapes of the opisthosomal annuli that may form various plates, protrusions, ridges and furrows.Based on these groups of characters, a higher classification and generic key of Eriophyoidea was developed by Amrine et al. [7] twenty years ago.Although this classification system is well designed for practical specialists and has been widely accepted by acarologists, it needs updating because many new supraspecific taxa of Eriophyoidea have been established, and progress in finding new morphological characters and understanding eriophyoid anatomy has been made since then [13][14][15][16].Additionally, molecular phylogenetic studies of the last decade indicate that the current system of Eriophyoidea does not reflect the phylogeny and that many supraspecific taxa defined by morphology are artificial [8,14,[17][18][19].
The shape of setae is one of the characters used in the current systematics of gall mites.The shape of a certain seta is included as an obligatory trait in the diagnoses of some genera, but it does not discriminate any current suprageneric taxa [7].As a rule, setae are smooth, filiform and unbranched in Eriophyoidea, but in some taxa, some setae may be atypically shaped, e.g., bifurcated or angled.According to our estimations, in 24 genera, gnathosomal seta d is bifurcated.In slide-mounted specimens, the smaller branch (the one-directed laterad, Figure 1E) is very often broken.Because of this, many species that have bifurcated seta d were described as having it "angled" (Figure 2A), e.g., various species of Epitrimerus were transferred to Leipothrix after this artifact was discovered [20,21].When one branch of a bifurcated seta is rudimentary or completely suppressed, the seta also looks angled, e.g., pedipalp seta d in Leipothrix solidaginis Keifer [7] and the angled tarsal seta u in various Phyllocoptinae and Sierraphytoptinae species [22][23][24][25][26][27].
Leipothrix Keifer is the largest phyllocoptine genus, characterized by bifurcated pedipalp setae d, three longitudinal opisthosomal ridges, and the absence of femoral setae bv I and II [7].It comprises about 60 species, including a few with a taxonomic status that need confirmation because of unverified data on their chaetome [21].Sexual dimorphism, generally poorly pronounced in Eriophyoidea [2,28], is prominent in Leipothrix, since their males are usually twice as small and move notably faster than females (P.Chetverikov and J. Amrine unpublished observations).All Leipothrix spp.are vagrant, typically living on the lower leaf surface.A few of them have been reported as causing rust, discoloration, slight deformation, wrinkling of leaves and witches' broom, e.g., L. dipsacivagus (Petanović and Rector 2007) [29], but never causing true galls with a gall chamber (like a finger or pouch gall), erinea or bud galls that are characteristic of gall-forming Eriophyoidea [30,31].Most species (~98%) are known from the Palearctic and predominantly inhabit herbaceous plants.Only three species were described in the Southern Hemisphere: L. triquetra (Meyer 1990) and L. minidonta (Meyer 1990) from ferns in South Africa, and L. eichhorniae (Keifer 1979) from the invasive water plant Pontederia crassipes Mart. in Brazil.
Leipothrix has a wide range of phylogenetically remote hosts.About a quarter of Leipothrix spp.(15 spp., ~28%) are associated with early-derivative plant clades (ferns-4 spp., magnoliids-3, and monocots-8).The remaining species (39 spp., ~72%) occur on eudicots and most of them (29 spp.) inhabit asterids.A single species, Leipothrix juniperensis Xue and Yin 2020, is known from conifers.According to the original description [32], this species does not fit the diagnosis of Leipothrix because it has femoral setae bv I and II (absent in Leipothrix).Leipothrix Keifer is the largest phyllocoptine genus, characterized by bifurcated pedipalp setae d, three longitudinal opisthosomal ridges, and the absence of femoral setae bv I and II [7].It comprises about 60 species, including a few with a taxonomic status that need confirmation because of unverified data on their chaetome [21].Sexual dimorphism, generally poorly pronounced in Eriophyoidea [2,28], is prominent in Leipothrix, since their GenBank data on Leipothrix include 215 sequences (accessed on 1 August 2023).Among them, 213 sequences belong to Leipothrix sp. and L. juniperensis from China.They in-clude fragments of COX1, 18S and 28S genes and one complete mitochondrial genome (KX027362) [18].Two sequences belong to L. liroi (ITS1-5.8S,MH522408) from Primula sp. from Iran and Leipothrix sp.(D1D2 28S, KT070277) from the fern Cheilanthes viridis from South Africa [17].
In this paper, we aim to (1) assess the distribution of bifurcated and other atypicallyshaped setae across eriophyoid genera; (2) test the monophyly of the genus Leipothrix and the whole group of eriophyoid genera possessing such setae; (3) clarify the phylogenetic position of L. juniperensis via molecular markers; and (4) investigate the organization of the mitochondrial genome in Leipothrix in order to reveal if Leipothrix spp. shares a common mitochondrial gene order (MGO) and if this MGO deviates from those in other eriophyoid taxa.

Materials and Methods
In order to review the distribution of atypically shaped setae in Eriophyoidea, we performed an extensive literature search and screened original descriptions of eriophyoid taxa from our libraries and various well-illustrated regional and world catalogs containing morphological drawings of eriophyoids [7,[23][24][25][26][27]33,34].We also examined eriophyoid mites under light microscopy (LM) (differential interference contrast (DIC) and phase contrast (PC)), using a Leica DM2500 microscope, the slide-mounted eriophyoids from the Acarological Collection of ZIN RAS, focused mainly on the genera with atypically shaped setae.In order to investigate the behavior of eriophyoid setae under illumination of a blue laser (405 nm), we analyzed confocal laser scanning microscopy (CLSM) stacks of various eriophyoid taxa, obtained using a spectral confocal and multiphoton system Leica TCS SP2 with objectives 63× N.A. 1.4-0.60Oil lBL HCX PL APO and 40× N.A. 1.25-0.75Oil CS HCX PL APO, at an excitation wavelength of 405 nm, and an emission wavelength range of 415-750 nm, at 10-20% intensity from our previous studies [35][36][37][38].
For molecular studies, we obtained sequences of two genes (COX1 and D1D2 28S) of five phyllocoptines that have bifurcated setae d and complete mitochondrial genomes of four Leipothrix spp.(Table 1).For this purpose, we used the same methodology and protocols for DNA extraction, library preparation, PCR, genome sequencing, assembly and annotation as described in [39,40].Three sequence datasets (Cox1, 28S and mitogenomic) were made for molecular phylogenetic analyses.The Cox1 dataset included 1409 unique sequences of gall mites from GenBank.They were translated into amino acids and aligned in MAFFT [41,42] with default adjustments, resulting in the final alignment consisting of 423 amino acid positions.For creating the 28S dataset, we blasted sequence OR416172 of L. aegopodii against Eriophyidae and filtered the sequences of 45% coverage.The remaining 166 sequences were aligned and modified as described in [40].The mitogenomic dataset included 11 complete sequences of mitochondrial genomes, among them 7 sequences from Genbank [18,32,40,[43][44][45] and 4 sequences obtained in this study (Table 1).
Maximum likelihood analyses were conducted in IQ-TREE 2 [46].For gene evolution, the GTR + F + I + G4 model was selected for the 28S datasets and the mtART + R3 model was selected for the Cox1 dataset using ModelFinder [47], as implemented in IQ-TREE 2 based on the Bayesian Information Criterion.Branch support values were generated from the ultrafast bootstrap approximation (UFBoot), with 10,000 bootstrap alignments, 10,000 maximum iterations and a minimum correlation coefficient of 0.99.Values of a single branch test (SH-like approximate likelihood ratio test, SH-aLRT) with 10,000 replicates and ultrafast bootstrap support (UFBS) were labeled on the maximum likelihood (ML) trees.
For the mitogenomic analysis, mitochondrial rRNA (12S and 16S) and protein genes (translated into amino acids) were aligned using MAFFT, using an E-INS-I algorithm for 12S and 16S genes and a G-INS-i algorithm for the protein genes.The resulting alignments were modified using Gblocks [48,49], as described in [40].Sequences of the ATP8 gene were excluded because Gblocks failed to find reliable blocks in the alignment of this gene.For gene evolution, the GTR + F + I + G4 model was selected for the merged 12S + 16S datasets, mtZOA + F + G4 was selected for the merged ATP6 + COX2 + COX3 + NAD1 + NAD2 + NAD3 + NAD4 + NAD4L + NAD5 + NAD6 datasets, and the mtART + I + G4 model was selected for the merged COX1 + CYTB dataset using ModelFinder.All other steps of the analysis were similar to those described for the Cox1 and 28S analyses.Sequences of two phytoptid taxa (Fragariocoptes and Retracrus) from GenBank were used for rooting the trees.

Distribution of Atypically Shaped Setae in Taxa of Eriophyoidea
Except empodia and solenidia (not discussed in this paper), the setae in eriophyoid mites are usually unbranched and filiform [1,2,7].In ~50 genera from three families (Phytoptidae s.str., Eriophyidae s.str.and Diptilomiopidae), some setae are of an atypical shape: angled, bifurcated or with swellings (Figure 1, Table 2).These modifications are always in the basal part of the setae.Atypical setae are present mostly on the prosoma and include leg setae u , bv, ft and ft", gnathosomal setae d and v, and prodorsal shield setae ve and sc.The listed leg and gnathosomal setae may be angled or bifurcated (bearing an extra branch), whereas when prodorsal shield setae are modified, they have one or two bulb-like swellings near the base (Figure 1).A single phyllocoptine species (Leipothrix nagyi Ripka et al. 2020) has modified h2 [50], the paired setae that are located in the caudal part of opisthosoma in all Eriophyoidea [1].[50]).Angled gnathosomal v has been reported in a few genera of Diptilomiopidae (Figure 1F, Table 2).Drop-shaped setae and those with basal bulb-like swellings are known in sierraphytoptine genera Propilus (Figure 1H) and Retracrus (Figure 1I,J).No species with atypical setae have been registered in the conifer-associated lineage Nalepellidae except Nalepella sp., which has bulbous swellings near the bases of setae sc when observed under SEM (R. Petanović, personal communication, July 2011).
Among eriophyoid genera, there are a few in which atypical setae are present in some species and absent in others.For instance, in Notostrix, most species have no atypically shaped setae, a few species have angled u and in one species (N.trifida), bv and u are angled (Figure 1A-C).Leipothrix is the largest eriophyoid genus (~60 species), all members of which possess bifurcated d.Remarkably, at least seven Leipothrix spp.from our collections (L.aegopodiae, L. convallariae, L. femoralis, L. geranii, L. jaceae, L. knautiae, L. ranunculi, Leipothrix sp. and L. triquetra) have angled u , missed by previous authors (Figure 2G,H).Finally, most small and monotypic eriophyoid genera with atypical setae inhabit palms (Arecaceae) in South America and Africa and different subtropical dicotyledonous trees in Asia.

Microscopic Observations
Under a stereomicroscope (magnification about ×20-×80), the setae of eriophyoid mites are tiny hair-like structures, usually of distinct black color.This color is especially well seen in some Phytoptidae s.l.(e.g., in Nalepella and Novophytoptus with stout sc, in Oziella with long c1) and in various species of Eriophyidae s.l. that have long opisthosomal setae c2, d, h2.Some species of the listed phytoptid genera are capable of moving their long setae sc or c1 with an amplitude of up to ~50 degrees, apparently due to strong opisthosomal musculature operating the setal bases (I.G.Bagnjuk personal communication, 1996; P.E.Chetverikov, unpublished observations, 2004Chetverikov, unpublished observations, , 2020)).
Under conventional light microscopy (magnification ×1000), all common filiform setae in most slide-mounted specimens from our collections that we observed consist of two parts, a short proximal part and long distal part, often forming an obtuse angle that is very close to 180 • (Figure 3B,C).Because of this slight curvature, under CLSM adjusted to capture the reflected light of a laser, the reflection of the proximal and distal parts of a seta is different.Only one of these parts (usually the distal one, depending on the position of the specimen on the slide) reflects the light at the right angle to be captured by CLSM, whereas the other part does not produce reflection and is unobservable using the "reflection mode" of CLSM (Figure 3K,N).
In our collections, we have specimens of Pentasetacidae, Phytoptidae s.l. and Eriophyidae s.l., slide mounted under suboptimal conditions, allowing the dust of the air or other extraneous material to occur in the mounting medium.In these cases, the extraneous objects tend to form clusters in the form of dark drops attached to the curved area between the basal and distal parts of setae (Figure 3D-H).Under CLSM applied for capturing the emission light when illuminating mites with a blue laser (405 nm), the proximal part of all setae exhibits very strong autofluorescence, whereas the distal part produces no signal (Figure 3I,J,L,M).
Under conventional DIC LM and PC LM, the proximal and distal parts of eriophyoid setae differ by birefringence and thickness.The proximal (most basal) part always looks like a hollow, linear structure with dark outlines and lighter medial content that is green or blue depending on the applied objective.In long setae, especially in coxal setae 2a, genital setae 3a, and opisthosomal setae c2, d and f, the proximal part is often followed by a more or less distinct but always very tiny thickening (Figure 3A-C).It marks the curvature zone mentioned above and continues into a hair-like distal part that is dark and solid.Under SEM and LT-SEM, these tiny elements of setal morphology are rarely seen.They may be hidden under the layer of the sputter coating or they snap off because of the pressure changes.This may explain why bifurcating setae often end up looking angled under SEM.

Molecular Phylogenetics: Blast Searches and COX1 and 28S Analyses
Blast searches for D1D2 28S sequences of the four new sequences of Leipothrix (Table 1) against Eriophyidae returned, as the best hit, the sequence KT070277 of Leipothrix cf triquetra from Cheilanthes viridis from South Africa, with 99-100% coverage and 90-91% identity.Sequences of Leipothrix juniperensis were absent in the list of the 100 most similar 28S sequences returned by Blast for the four Leipothrix spp.
Blast searches for COX1 (MZ274920) and 28S (MZ289016) sequences of Leipothrix juniperensis except conspecific sequences returned various sequences of phyllocoptines associated with gymnosperms (e.g., Epitrimerus sabinae, Phyllocoptruta platycladusa, Stenacis thunbergii), all of them without bifurcated setae.Inclusion of the new COX1 and 28S sequences of the four Leipothrix spp. in the Blast searches did not change the result for L. juniperensis.

Mitogenomics
Four new sequences of the complete mitochondrial genomes of Leipothrix aegopodii, L. convallariae, L. knautiae, and Leipothrix sp.A were assembled and annotated (Figure 5, Table 3).The average size of a mitogenome is 13,593 ± 110 bp.Each mitogenome includes 37 similarly ordered genes (13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region), 10 of which are located on the negative chain.Protein-coding genes terminate with stop codons TAA (67.31%) or TAG (26.92%), except genes NAD3 (in L. knautiae and L. convallariae) and NAD5 (in L. convallariae), which terminate with mononucleotide T. The control region (CR) in all mitogenomes is flanked by genes trnL and NAD2 and in size from 38 bp in L. convallariae to 184 bp in L. knautiae (Table 3).In Leipothrix, sp.A and L. knautiae the CR has complementary poly-G and poly-C fragments, forming a large D-loop of ~100 bp.The four new mitogenomes of Leipothrix spp.comprise the same constant blocks I, II, III and variable zones A,B,C (Figure 5), recently defined in other published mitogenomes of Eriophyidae [18,32,40,[43][44][45].They share the following unique traits: (1) the trnK gene precedes the COX1 gene, (2) two tRNA genes coding leucine are located on different chains of mitochondrial DNA, (3) a cluster of tRNA genes W-V precedes the 12s rRNA gene, (4) genes 16s rRNA and COX1 flank a group of uniquely arranged genes and the control region Y-L-(CR)-NAD2-Q-C-M-K (genes located on the negative chain are underlined).The only mitogenome in GenBank assigned to the genus Leipothrix (KX027362.1,L. juniperensis) [18] does not have the traits listed above (Figure 5).It has trnK flanked by COX2 and trnD, both trnL genes located on the negative chain, genes trnV and trnW flanking the 16s rRNA gene, and a gene cluster W-NAD2-M-C lacking a CR and situated between its 16s rRNA and COX1 genes.The MGO in this species is closest to that in Phyllocoptes taishanensis (NC_029209) [43], except the position of the trnQ gene and the number and position of the CR: a single CR between trnY and 12S rRNA in P. taishanensis vs. three CR located in zones A, III and C in L. juniperensis (Figure 5).
Maximum likelihood analysis of the mitogenomic dataset produced a poorly resolved tree, comprising two clades-X and Y (Figure 5).The "true Leipothrix" is monophyletic.Leipothrix juniperensis and tL are nested within clades X and Y (correspondingly).

Discussion
Is setal bifurcation an ancestral character state in Eriophyoidea?Our observations indicate the following: (a) common filiform setae in Eriophyoidea are usually slightly bent near the base and may be thickened, which is only noticeable with high-level optics; (b) dust particles and other extraneous material occurring in the mounting medium tend to form clusters around this swelling; and (c) proximal and distal parts of setae differ in light reflection and autofluorescence.We also showed that in Eriophyoidea, all modifications always happen in the basal part of a seta and they are scattered throughout different phylogenetic lineages of Eriophyoidea.These data suggest that eriophyoid setae are more complex than previously thought.We hypothesize that ancestrally, all setae in eriophyoid mites were bifurcated and later simplified into filiform setae, with one of the two setal branches shortened or completely reduced, the latter resulting in an angled seta.This "simplification" scenario agrees with the general reduction trend in Eriophyoidea.It is also more parsimonious than the alternative scenario, which would require that in many phylogenetically unrelated lineages, the simple filiform setae have transformed into bifurcated or angled setae in parallel.Non-monophyly of the groups of eriophyoid taxa possessing different, atypically shaped setae has been revealed by COX1 and 28S molecular phylogenetics (Figure 4) and other analyses that include larger datasets [8,18,19].This agrees with the hypothesis on the plesiomorphic nature of bifurcated setae in Eriophyoidea.
Synapomorphic status of bifurcating setae.Based on a consensus of molecular and morphological phylogenetic analyses, there is now very strong support for a close relationship between Eriophyoidea and Nematalycidae [4,5,44,[51][52][53].The bifurcating form of setae in Eriophyoidea provides additional evidence for this affiliation.Bifurcating and trifurcating setae are rare and unusual structures in Acariformes.The vast majority of mite setae are either unbranched or have more than three branches (hyper-furcating).But in Nematalycidae, almost all setae are unbranched, bifurcating or trifurcating [54][55][56][57][58].If bifurcated setae are the ancestral condition in Eriophyoidea, hypo-furcating setae (herein defined as setae with two or three branches) represent another potentially important synapomorphy that unites Eriophyoidea with some or all Nematalycidae.
Typology and evolution of furcating setae.The hypo-furcating form of setae in Nematalycidae and Eriophyoidea appears to represent a derived form that is entirely distinct from the hyper-furcating form that occurs in Trombidiformes and most lineages within Endeostigmata (a basal grade that is probably paraphyletic to Trombidiformes, Oribatida and Eriophyoidea [34,52,53]).By far the most common form of hyper-furcating seta is a serial branching (seri-furcating) seta, in which the branches form rows along a single, central stem (Figure 6A-D).Fractal or dendritic (tree-like) branching (dendro-furcating) represents another form of branching seta, in which branches subdivide into further branches [59].However, that type of seta is very rare in mites.In some Endeostigmata, the stems of seri-furcating setae on the hysterosoma often swell distally to form bulbous, club-or wedgeshaped structures [5,59,60], whereas in Trombidiformes, the stems of seri-furcating setae are usually filiform [3].Due to its dominance among basal families of Endeostigmata [61], the seri-furcating seta likely represents the plesiomorphic form of seta for Acariformes (Figure 6A).In addition to the unbranched form of seta, seri-furcating setae are also very common in Trombidiformes [62][63][64][65].Whereas in some species of Trombidiformes the setules (branches) of seri-furcating setae are long, e.g., in Allothrombium fuliginosum (Hermann) [66] (Figure 338E), in others, the setules are so short so as to be vestigial and almost indiscernible, e.g., in Abrolophus rubipes Trouessart [66] (Figure 338F).Hypo-furcating setae are extremely rare in Trombidiformes.The relatively common unbranched form of seta in this lineage (e.g., Metatarsonemus [67]) is readily explained by the parallel reduction and eventual loss of the many setules of seri-furcating setae (Figure 6A-E), and so setae with extremely short setules (Figure 6D) appear to represent a transitional form between setae with long setules (Figure 6A) and unbranched setae (Figure 6E).Notably, some trombidiform species simultaneously bear smooth and unbranched setae in addition to seri-furcating setae with extremely short setules [67].Due to its dominance among basal families of Endeostigmata [61], the seri-furcating seta likely represents the plesiomorphic form of seta for Acariformes (Figure 6A).In addition to the unbranched form of seta, seri-furcating setae are also very common in Trombidiformes [62][63][64][65].Whereas in some species of Trombidiformes the setules (branches) of seri-furcating setae are long, e.g., in Allothrombium fuliginosum (Hermann) [66] (Figure 338E), in others, the setules are so short so as to be vestigial and almost indiscernible, e.g., in Abrolophus rubipes Trouessart [66] (Figure 338F).Hypo-furcating setae are extremely rare in Trombidiformes.The relatively common unbranched form of seta in this lineage (e.g., Metatarsonemus [67]) is readily explained by the parallel reduction and eventual loss of the many setules of seri-furcating setae (Figure 6A-E), and so setae with extremely short setules (Figure 6D) appear to represent a transitional form between setae with long setules (Figure 6A) and unbranched setae (Figure 6E).Notably, some trombidiform species simultaneously bear smooth and unbranched setae in addition to seri-furcating setae with extremely short setules [67].
Some endeostigmatids, such as Micropsammidae, have seri-furcating setae with a low number of setules (Figure 6F), whereas the trifurcating, bifurcating and unbranched setae of Nematalycidae and Eriophyoidea have too few branches to be seri-furcating.Due to this absence of seri-furcating setae, the unbranched setae of eriophyoids and nematalycids cannot be readily explained by the parallel reduction in many setules.Instead, individual setules have probably been reduced and lost sequentially (Figure 6F-I), such that the number of setules gradually diminishes until it reaches zero, resulting in an unbranched seta (Figure 6I).In some or perhaps all cases, individual setules may have gradually diminished in length, so that only a single vestigial stump or projection remains (Figure 6H) before any trace of a setule is completely gone (Figure 6I).In Nematalycidae and Eriophyoidea, bifurcating setae are often observed that have this stump-like vestige of a setule (Figure 6H).These setae are referred to as semi-bifurcating in the description of the nematalycid, Osperalycus tenerphagus Bolton and Klompen [57].The exact same type of setae is especially abundant on the leg segments of the nematalycid, Psammolycus delamarei Schubart [58].
Therefore, the unbranched form of seta in eriophyoids and nematalycids appears to have arisen in a completely different way from the unbranched form of seta in trombidiform mites.Moreover, the absence of seri-furcation and the presence of hypo-furcation in both Eriophyoidea and Nematalycidae further weakens the case for the placement of Eriophyoidea within Trombidiformes.
Taxonomic status of Leipothrix juniperensis and monophyly of Leipothrix.Recently, Yin et al. [19] examined the accuracy of molecular delimitation methods (BIN, ABGD, ASAP, GMYC and mPTP) and advocated for employing multiple analytical approaches to aid correct species delimitation in gall mites.A priori, the effectiveness of these methods depends on the availability of the carefully curated sequences that are stored in public databases (e.g., GenBank), which have unique numbers and are assigned to a peer-reviewed paper verifying the origin of the sequences [68].With new submissions, the number of erroneous sequences of Eriophyoidea uploaded to GenBank has been increasing every year [40], which makes it difficult to obtain correct molecular cladograms using data from this database.For instance, in this study, we found that the sequence MW251739 of a gall mite Acalitus vaccinii belongs to a crustacean (blastx 99.4% similarity with QIZ03131 of Campylaspis sulcata [69]), and eriophyoid sequences MZ483068 of Leipothrix sp. 1 XFX-2017 [18] and KM111096 of Cheiracus sulcatus [70] are 100% identical, meaning a wrong generic assignment.
Four GenBank sequences (MZ255376, MZ274920, MZ289016, MZ326598) are assigned to Leipothrix juniperensis Xue and Yin, 2020 in [32].It was described from the samples containing specimens of Epitrimerus sabinae s.l.Xue and Hong 2005 and collected from Juniperus chinensis L. (Cupressaceae) from various locations in China.The morphological concept of L. juniperensis was tested using molecular methods, including DNA-based species delimitation, phylogenetics, haplotype network and comparative mitogenomics [32].Yet, according to the original description, it does not fit the morphological diagnosis of Leipothrix.
Among eriophyoid genera, "juniperensis" is morphologically closest to Cereusacarus Xue et al., also described from China [71].They share the bifurcated pedipalp seta d, legs and opisthosoma with usual series of setae (including bv I and II present, contrary to Leipothrix, in which bv are absent), as well as opisthosoma with the middorsal ridge ending before the lateral ridges.However, "juniperensis" differs from Cereusacarus in some body shape characteristics.In Cereusacarus, the middorsal opisthosomal ridge ends in a furrow, the first five dorsal annuli are almost as wide as the prodorsal shield, with the next annuli abruptly narrower, and the dorsal and ventral annuli are not differentiated [71].In the protogyne of "juniperensis", it is uncertain whether the middorsal opisthosomal ridge ends in a furrow.The annuli taper gradually from the prodorsal shield posteriad, and the opisthosomal annuli are differentiated into broader dorsal semi-annuli and narrower ventral semi-annuli [32].Since, morphologically, "juniperensis" much more closely resembles Cereusacarus than Leipothrix, we exclude it from Leipothrix and provisionally transfer it to Cereusacarus: C. juniperensis (Xue and Yin, 2020 in [32]) comb.nov.It should be noted that sequences of Cereusacarus and "juniperensis" do not cluster together in our COX1 tree (Figure 4).Therefore, the proper generic placement of "juniperensis" needs further testing.
Overall, our study indicates the importance of carefully examining the chaetome of eriophyoid mites for correct generic assignments and calls for the curation of sequences after they have been uploaded to GenBank.It also points in several new directions that would contribute to a further understanding of the phenomenon of atypically shaped setae in Eriophyoidea and their evolution: (1) reexamination of old eriophyoid taxa in order to reveal the true morphology of their setae; (2) comparative studies of the chaetome of Eriophyoidea and Nematalycidae, including additional investigation of the fine structure of their setae with the aid of various microscopic techniques, including transmission electron microscopy; and (3) taxonomic revisions, molecular phylogenetics and mitogenomics of eriophyoid genera with atypically shaped setae, especially palm associated genera, e.g., Notostrix, Propilus, Tumescoptes and Retracrus.

21 Figure 5 .
Figure 5. Maximum likelihood phylogeny (12 mitochondrial protein genes, 12S, and 16S) of Eriophyoidea (left) and gene orders in the mitochondrial genomes included in the analysis (right).New sequences of Leipothrix spp.("true Leipothrix", tL) are colorized red and L. juniperensis ("false Leipothrix", fL) is colorized blue.Branch labels are the following: SH-aLRT support (%)/ultrafast bootstrap support (UFBS, %).Black circles (•) indicate taxa with bifurcated pedipalp seta d.The constant blocks (I-III) and variable zone (A-C) of mitochondrial genes in eleven eriophyoid mite species are indicated.Translocation of the trnK gene from zone A to zone C is shown by the pink arc-shaped arrow.Black arrowheads point to the position of control regions.Genes located on the negative chain of mitochondrial DNA are underlined.Notations: cx-Cytochrome c oxidase (green), atp-ATP synthase (orange), nd-NADH dehydrogenase (blue), cyb-Cytochrome b (purple), 12S and 16S-rRNA genes (red and yellow), X and Y-two clades of Eriophyidae s.l.recovered in this analysis.

Figure 5 .
Figure 5. Maximum likelihood phylogeny (12 mitochondrial protein genes, 12S, and 16S) of Eriophyoidea (left) and gene orders in the mitochondrial genomes included in the analysis (right).New sequences of Leipothrix spp.("true Leipothrix", tL) are colorized red and L. juniperensis ("false Leipothrix", fL) is colorized blue.Branch labels are the following: SH-aLRT support (%)/ultrafast bootstrap support (UFBS, %).Black circles (•) indicate taxa with bifurcated pedipalp seta d.The constant blocks (I-III) and variable zone (A-C) of mitochondrial genes in eleven eriophyoid mite species are indicated.Translocation of the trnK gene from zone A to zone C is shown by the pink arc-shaped arrow.Black arrowheads point to the position of control regions.Genes located on the negative chain of mitochondrial DNA are underlined.Notations: cx-Cytochrome c oxidase (green), atp-ATP synthase (orange), nd-NADH dehydrogenase (blue), cyb-Cytochrome b (purple), 12S and 16S-rRNA genes (red and yellow), X and Y-two clades of Eriophyidae s.l.recovered in this analysis.

Table 1 .
Collecting data and GenBank accession numbers for five eriophyoid mite species.

Table 3 .
Characteristics of mitochondrial genomes of four Leipothrix spp.Notations: sp1-Leipothrix aegopodii, sp2-Leipothrix sp.A, sp3-L.knautiae, sp4-L.convallariae, J-positive chain of mitochondrial DNA, N-negative chain of mitochondrial DNA.In the first column, the codons are given in brackets after each corresponding tRNA.Numbers in brackets indicate the amounts of overlapping nucleotides between adjoining genes (the minus indicates genes located on the negative chain).

Table 3 .
Characteristics of mitochondrial genomes of four Leipothrix spp.Notations: sp1-Leipothrix aegopodii, sp2-Leipothrix sp.A, sp3-L.knautiae, sp4-L.convallariae, J-positive chain of mitochondrial DNA, N-negative chain of mitochondrial DNA.In the first column, the codons are given in brackets after each corresponding tRNA.Numbers in brackets indicate the amounts of overlapping nucleotides between adjoining genes (the minus indicates genes located on the negative chain).